[Human plasma fibronectin. Comparison of methods for preparation of a concentrate for therapeutic use from different sources].
Three methods, successive precipitations, affinity chromatography on immobilized gelatin and immunoaffinity chromatography with monoclonal anti-fibronectin antibodies were optimized and compared in order to be used for large scale preparation of human plasma fibronectin (Fn). The functional properties of the various Fn preparations were investigated by means of two assays: quantitation of the gelatin-binding activity by ELISA and quantitation of the Fn-mediated attachment of fibroblasts on plastic. Functional alterations of the purified Fn were observed when it was isolated by successive precipitations. Both chromatographic methods provide a rapid and convenient way for isolation of pure and functional Fn. Mass production of monoclonal antibodies is too expensive and legislative requirements for the therapeutic use of monoclonal antibodies are limiting factors for the choice of immunopurification as large scale isolation procedure. Plasma Fn can be isolated from different sources: fresh frozen plasma, cryoprecipitate supernatant or by-products from factor VIII preparation. When gelatin-Sepharose chromatography is performed under optimized conditions, fibronectins isolated from these sources show similar properties. Large scale purifications of Fn from a by-product of factor VIII preparation were performed either by gelatin affinity chromatography or by successive precipitations. These two purification methods can be easily scaled-up since the data obtained closely correlate with analytical results. The chromatographic method supplies a higher purified (98 vs 75%) and functional (95 vs 50%) material when compared with successive precipitations. Yield is also higher (50 vs 26%). The starting material undergoes viral inactivation and the affinity purified Fn, sterile, atoxic, apyrogen, which can be freeze-dried without additives fulfils all requirements for an injectable product.
Regnault
V
V
INSERM U 284, Plateau de Brabois, Vandoeuvre-lès-Nancy.
Rivat
C
C
Geschier
C
C
Stoltz
J F
JF
D000911:Antibodies, Monoclonal / Q000276:immunology; D002448:Cell Adhesion / Q000187:drug effects; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D003080:Cold Temperature; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects; D005353:Fibronectins / Q000097:blood / Q000302:isolation & purification* / Q000494:pharmacology; D007163:Immunosorbent Techniques; D010865:Pilot Projects; D010949:Plasma / Q000737:chemistry
D000911:Antibodies, Monoclonal; D005353:Fibronectins
Fibronectine plasmatique humaine. Comparaison de méthodes de préparation d'un concentré à usage thérapeutique à partir de différentes sources.